The overall aim of the proposed research is to quantify the spatio-temporal variation of cytoplasmic [Ca2+] during excitation-contration coupling in mammalian heart cells, in order to understand the cellular processes that produce the cytoplasmic [Ca2+] transient, and to quantify the influence of Ca2+ on cellular functions. To be tested in the present research is an hypothesis which comprises a specific aspect of the overall aim. Hypothesis: Ca2+ entering the cell via surface membrane Ca2+ channels enters rapidly into junctional regions of the sarcoplasmic reticulum (SR), rather than accumulatilng in the cytoplasm. In Purkinje fibers, release of Ca2+ into the cytoplasm from junctional SR underlies a rapid initial rise in cytoplasmic [Ca2+] (L1 component of the aequorin signal). Ca2+ diffuses to regions of SR deep in the cell, triggering Ca2+ release (component L2). In ventricular cells, separate components of Ca2+ indicator signals are not evident because the diffusion delays are small, compared to Purkinje fibers. Predictions and tests of this hypothesis are: 1) In Purkinje fibers, L1 is highly spatially localized. To test this prediction, the spatial variation of the cytoplasmic [Ca2+] transient will be assessed by the extensive use of different intracellular Ca2+ indicators which give different estimates of [Ca2+] when spatial gradients of [Ca2+] are present. 2) L1 should depend on Ca2+ loading of junctional SR. A signal arising directly from entering Ca2+, and not from Ca2+ released from SR, will be sought by pulsing high [Ca2+] solution onto single, isolated, Ca2+ depleted heart cells which contain a Ca2+ indicator. 3) The release of Ca2+ from deep SR, signalled by L2, should depend on L1 and not directly on membrane depolarization. To test this prediction, [Ca2+] transients will be studied in voltage-clamped, aequorin containing Purkinje fibers, and in fibers containing a Ca2+ buffer, quin2. (Quin2 will bind Ca2+ released from junctional SR; no other Ca2+ release should then occur). This research will reveal the extent of spatial gradients of [Ca2+] during E-C coupling and it will elucidate differences in E-C coupling between Purkinje fibers and ventricular tissue. It will permit determination of the roles of entering Ca2+ and Ca2+ induced Ca2+ release from the SR in intact tissue.